Lipoxygenase (LOX) enzymes are useful in the oxidation of fatty acids for a variety of commercial purposes. Peroxidases (POD) are oxidoreductases with many applications in medical, environmental and industrial processes including removing aromatic amines or phenols from water by peroxidase-catalyzed transformation (Klibanov, A. M. and Morris, E. D. [1981] Enzyme Microb Technol. 3:119; Klibanov, A. M. et al. [1983] Science 221:259). Immobilized peroxidases have been used in biosensors to detect pesticide residues (Sandberg, R. G. et al. [1992] Biosensor design and application, Mathewson, P. R. and Finley, J. W. eds., ACS, Washington D.C., pp. 81-88).
Labile proteins such as lipoxygenases, peroxidases and lipases lose most of their activity in aqueous solutions quickly. Immobilizing the enzyme would enable a continuous process that gives high substrate conversions, good product recovery, and minimal loss of enzyme activity. Conventional methods of enzyme immobilization include covalently binding or adsorbing the enzymes onto a solid support. LOX has been immobilized by adsorption to glutenin, gliadin, glass wool, talc, polymer beads, and ion-exchange supports (Cuperus, F. P. et al. [1995] Catalysis Today 25:441-445; Battu, S. et al. [1994] J. Agric. Food Chem. 42:2115-2119). The matrices used in covalent binding of LOX include oxirane acrylic beads, CNBr-activated sepharose and agarose, and carbonyidi-imidazole-activated polymer (Parra-Diaz, D. et al. [1993] Biotech. Appl. Biochem. 18:359-367). Although improving the stability of the enzyme, covalent and ionic bonds formed by these methods can cause a decrease in enzyme activity. For example, the adsorption of S. tuberosum lipoxygenase on talc retained only 53% of its activity in immobilized form (Battu et al., supra). Immobilization of enzymes by entrapment has been achieved by encapsulating enzymes through sol-gel processes (Avnir, D. et al. [1994] Chem. Mater. 6:1605-1614). The entrapped enzymes retained much of their activity and had better stability in the sol-gel matrices. Extension of this technique, however, was limited by two shortcomings of sol-gel materials: their brittleness and narrow pore network (Heichal-Segal et al. [1995] Biotechnology 13:798-800). Efforts were made to improve the activity of immobilized enzymes by introducing matrix-relaxing additives, such as algenate or polymers (Heichal-Segal et al., supra; Shtelzer, S. et al. [1992] Biotech. Appl. Biochem. 15:227-235) into sol-gel matrices, or mixing alkyl-substituted silanes in a specific ratio (Reetz, M. T. et al. [1996] Biotechnol. Bioengineering 49:527-534). Despite these improvements, however, efficient alternative methods are still needed for enzyme immobilization to provide high activity and increased storage stability. Most methods for immobilizing LOX provide materials that are not stable longer than about a month at room temperature. The best immobilization methods in the literature, based on the covalent binding or adsorption of lipoxygenases, typically immobilize lipoxygenases to 70% of protein content with about 50% retainment of enzyme activity.
Clay minerals are naturally occurring phyllosilicates (i.e., layered silicates) with good intercalative properties. Because their layered structures can be broken down into nanoscale building blocks, phyllosilicates can serve as a framework for intercalation. Metal hydroxyl polymeric cations, alkylammonium ions, polymers, and their combinations have been intercalated into phyllosilicates to form a broad spectrum of materials ranging from pillared clays and organoclay, to polymer-clay nanocomposites. The intercalated phyllosilicates exhibit good mechanical and thermal stability, controlled pore size (0.2-1 .mu.m) and ion mobility, and high adsorption capacity. (Monnier, A. et al. [1993] Science 261:1299-1303; Pinnavia, T. J. [1983] Science 220:365-371; Vaia, R. A. et al. [1994] Chem. Mater. 6:1017-1022; Yan, Y. and Bien, T. [1993] Chem. Mater. 5:905-907; and Burnside, S. D. and Giannelis, E. P. [1995] Chem. Mater. 7:1597-1600.)
Compositions providing highly active immobilized bioactive proteins which are storage-stable are needed, as are efficient methods for producing such compositions.
All publications referred to herein are incorporated by reference to the extent not inconsistent herewith.